Summary: Many chemotherapeutics kill cancer cells by inducing DNA damage interfering with DNA replication.ReplicationforkscanreversetoaidtherepairofDNAdamageinducedbychemotherapeutics,and BRCA proteins are key to protecting the reversed forks from nucleolytic degradation. In absence of BRCA, reversed replication forks are extensively degraded by nucleases, leading to chemosensitivity. Moreover, chemoresistancehasbeenlinkedtotherestoredabilityofBRCA-deficientcellstoprotectforksfromdegradation through mechanisms that remain unclear. Thus, understanding how cells protect stalled DNA replication forks andpromotetheirrecoveryiscriticallyimportantfordevelopingandimprovingstrategiesforcancertherapy.In thisapplication,wecombinetheexpertisefromtheZouandVindignilaboratoriestoinvestigatethemechanisms of DNA replication fork protection and recovery in BRCA1-proficient (Aim 1) and -deficient (Aim 2) cells. The ZoulabhasextensiveexperienceintheATRsignalingpathway,whichiscrucialforstabilizingthegenomeduring DNAreplication.TheVindignilabhasdiscoveredprincipalstepsoftheforkreversalpathway.Workingtogether, we have uncovered a number of new players involved in fork protection. In the preliminary studies leading to Aim 1, we found that ATRplaysa previouslyunrecognized role inprotecting stalled/reversed replication forks from nucleolytic degradation in BRCA1-proficient cells. We also showed that ATR is required for the efficient recoveryofstalledforks.Basedonthispremise,wehypothesizethatATRactslocallyatstalledreplicationforks toprotectreversedforksfromnucleolyticdegradationandtopromotetheirrestartafterdrugremoval.Aim1will determine the mechanismsby which ATRprotects stalled/reversedforksandpromotes fork restartin BRCA1- proficientcells.InthepreliminarystudiesleadingtoAim2,wehaveinvestigatedhowstalledforksareprotected andhowtheyrecoverincellslackingBRCA1.WefoundthatextensivelydegradedreplicationforksinBRCA1- deficient cells can recover through a pathway mediated by Rad18 and Ubc13. Furthermore, when BRCA1- deficientcellsacquirePARPinhibitorresistance,forkprotectionisrestoredviaaPALB2-dependentmechanism, which reliesonATR activity. Notably, both Ubc13 and PALB2 are functionally linked tothe E3 ubiquitin ligase RNF168,raisingthepossibilitythatUbc13,RNF168,andPALB2mayactinthesameaxistoprotectstalledforks and promote fork recovery independently of BRCA1. We hypothesize that both Ubc13 and PALB2 have unanticipated roles in fork recovery/protection in the absence of BRCA1, and their functions may be linked by RNF168. Aim2 will investigatehow Ubc13promotes fork recovery inBRCA1-deficient cells, howPALB2and ATR protect stalled forks in BRCA1-deficient PARP inhibitor-resistant cells, and whether a Ubc13-RNF168- PALB2axispromotesbothforkprotectionandforkrecoveryintheabsenceofBRCA1.Collectively,thesestudies willtransformcurrentmodelsofforkstabilizationandrecoveryinBRCA1-proficientand-deficientcells,providing amechanisticbasisfornewtherapeuticstrategiesthatexploitthereplicationstressincancercells.